Does Probiotic Consumption Enhance Wound Healing? A Systematic Review

The use of probiotics is one of the emerging lines of treatment for wound healing. This systematic review aimed to summarize currently available evidence on the effect of oral or enteral probiotic therapy on skin or oral mucosal wound healing in humans. To verify the developments in this field and the level of available scientific evidence, we applied a broad search strategy with no restrictions on wound type, target population, probiotic strain, or intervention protocol used. This review included seven studies involving 348 individuals. Four studies reported positive outcomes for healing improvement after probiotic therapy, and none of the studies reported adverse effects or a marked increase in wound healing time. The positive or neutral results observed do not generate strong evidence regarding the effectiveness of probiotics for wound healing. However, they suggest a promising field for future clinical research where the probiotic strains used, type of wounds, and target population are controlled for.


Introduction
Skin and mucosal wounds encompass a wide variety of injuries, including a surgical scar, pressure ulcer, extensive burn, and an open abdominal wound. Healing is an inherent process in all wounds, regardless of the cause. It is a systemic, dynamic, and expected process related to the general conditions of the organism [1], and can be negatively or positively influenced by local and systemic factors. The local factors that hinder wound healing include ischemia, infection, surgical technique, foreign body, and oedema [2], while systemic factors include vitamin deficiencies, malnutrition [3,4], and other conditions such as diabetes mellitus [5] and cardiovascular and respiratory diseases [1].
Wound care practices involve technical procedures, topical agents, and dressings, as well as holistic and systemic treatment, where the patient, and not just the wound, are observed [6]. An emerging treatment line for skin wounds and conditions is the use of probiotics, defined by the International Scientific Association of Probiotics and Prebiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [7].
Probiotics have been extensively investigated, and their role in improving infections and intestinal healing is well-known [8,9]. Researchers have reported that probiotics may have other health-promoting effects beyond intestinal wellbeing [10][11][12], such as preventing recurrent urinary tract infections in women and reducing respiratory tract infections [13][14][15]. In addition, a narrative literature review reported the beneficial effects of oral administration and topical application of probiotics for the treatment of skin diseases [15].
Studies have been conducted in order to evaluate the use of probiotics to enhance wound healing. Yu et al. reviewed the usefulness of oral and topical probiotics for certain dermatological diseases. The authors suggested that these interventions could be effective in the treatment of certain inflammatory skin diseases, with a promising role in promoting wound healing and managing skin cancer [16]. However, the use of probiotics as a nutritional supplement to treat skin or mucosal wounds was not emphasized in their review [16]. Animal studies have demonstrated a positive effect of probiotics in reducing bacterial load and increasing tissue repair [17,18]. In addition, in vitro studies [8,19] have demonstrated a positive effect for probiotics on the recovery of structural elements of the skin.
Although in vitro and in vivo animal studies support the potential for probiotics to promote skin healing, it is of paramount importance to investigate the level of existing evidence reported in human studies. Therefore, the aim of this systematic review was to summarize currently available evidence on the effect of oral or enteral probiotic therapy on skin or oral mucosal wound healing in humans.

Protocol and Registration
This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [20] and the protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) under the registration number CRD42020150682.

Eligibility Criteria
Randomised and non-randomised placebo-controlled trials that recruited people of all age groups and both sexes, with skin or oral mucosal wounds, receiving oral or enteral probiotic therapy with or without antibiotic treatment, and with healing assessment data, were included. Given that the preliminary searches identified few studies when limited to the type of wound, probiotic, or target population, we applied a broad search strategy to verify the scientific evidence level and evaluate the developments in this field. Reviews, congress abstracts, chapters of books, meeting proceedings, and studies without clear outcomes were not included. Furthermore, animal studies, in vitro studies, and studies with topical use of probiotics were excluded. There were no restrictions on the date, language, or publication status.

Information Sources and Search Strategies
The search strategy was developed based on the criteria recommended by the Peer Review of Electronic Search Strategies (PRESS) checklist [21] and submitted for revision by a researcher experienced in conducting systematic reviews. Detailed search strategies with adaptations of descriptors and term combinations were elaborated on according to the specificities of the following databases: Medline (through PubMed), Embase, Lilacs, Scopus, and Web of Science. A partial grey literature search was also conducted using the ProQuest database of theses and dissertations and Google Scholar, wherein the search was limited to the first 200 articles found. Finally, a search was performed of the clinical trials record database ClinicalTrials.gov (Table S1).
Rayyan QCRI software (Qatar Computing Research Institute ® , Doha, Qatar) [22] was used to remove duplicates and facilitate screening of the identified records. All references were managed using Mendeley Desktop software (Version 1.19.8; Mendeley Ltd., London, UK).

Study Selection
The study selection process was conducted in two phases by two independent researchers. In phase one, articles were selected according to their titles and abstracts based on the inclusion criteria. Any disagreements were resolved by consensus. In phase two, the selected articles were read in their entirety, and those that met the inclusion criteria were included. A manual search of the reference lists of the selected articles was also performed.

Data Collection Process
Data extraction was also performed independently by two authors. Any disagreements were resolved by consensus. The following data were extracted from the selected articles and recorded in an electronic spreadsheet: country, aim and study design, subjects/patients, type of wounds, intervention protocol, healing assessments, outcome of interest, possible adverse effects of probiotic therapy, and whether probiotic therapy improved wound healing.

Risk of Bias in Individual Studies
The critical appraisal tools recommended by the Joanna Briggs Institute [23] for randomised controlled trials and quasi-experimental studies (non-randomised experimental studies) were used to assess the risk of bias of the included studies. Two reviewers independently evaluated each study, and a third reviewer resolved disagreements. Both tools have the answers 'yes', 'no', 'unclear', or 'not applicable' for the questions. For this review, when all answers were 'yes', the study was classified as having a low risk of bias, and if any answer was 'no' or 'unclear', the study was classified as having a risk of bias. No scores were assigned; the results for each question were expressed as the frequency of each classification. In the cases where information was not clear, at least two attempts were made to request additional information from the authors. Evaluation of the risk of bias was not used as a part of the eligibility criteria for article inclusion.

Synthesis of Results
The primary outcome investigated was wound healing after probiotic therapy, and the secondary outcome was the safety of probiotic consumption. To assess the primary outcome, the methods of healing assessments used in each eligible study were examined. The quantitative or qualitative methods used to evaluate the outcome were identified. For the assessment of the secondary outcome, the reported adverse effects in the studies were considered.

Study Characteristics
The healing assessments were conducted locally based either on the need for grafting [26], number of dressing changes per day and post-operative wound complications [27], wound duration and operative days for excision and graft [28], ulcer mean surface area and volume [29], mean body surface area grafted and mean graft loss [24], and healing clinical scores [25,30].

Risk of Bias within Studies
Three of the seven studies included in this review [26,29,30] had a low risk of bias. Six parameters of the critical appraisal checklist were met in all randomised controlled clinical trials [25][26][27][28][29][30] (Figure 2). The only non-randomised clinical trial [24] that was included was found to meet seven of the nine parameters assessed using the instrument (Table S3).

Results of Individual Studies
El-Ghazely et al. found that paediatric patients undergoing probiotic therapy had a significantly lower need for grafting; when grafting was not performed, there was a significant decrease in the time required for complete healing of the wound [26]. Tahir et al. found that adult patients receiving probiotic therapy had a larger mean grafted body surface area than those in the control group, but not significantly so. In the same study, the mean graft loss was significantly higher in the control group [24]. In contrast, Mayes et al. did not report differences in the number of operative days for excision and grafting procedures, or the time needed for healing in paediatric patients with or without probiotic therapy [28].
Positive results were also observed for probiotic treatment of surgical wounds [27] and diabetic foot ulcers [29]. Esposito et al. found that the daily frequency of dressing changes was significantly lower in paediatric patients on probiotic therapy (1.7 times/day) than in those in the antibiotics group (3.3 times/day) and in the antibiotics and placebo group (2.8 times/day). In parallel, the incidence of post-operative wound complications was significantly higher in the groups without probiotic therapy [27]. Mohseni et al. observed a significant improvement in the ulcer healing parameters with respect to its length, width, and depth in adults on probiotic therapy [29]. Three studies, two among adults [25,30] and one among paediatric patients [28], did not find improvement in wound healing with probiotic therapy.
It was not possible to conduct a meta-analysis due to the different outcomes assessed across the studies, mainly because of the wide variation in the methods used to evaluate the effects of probiotic therapy, population heterogeneity, type of wounds, and intervention protocols of each study.

0%
20% 40% 60% 80% 100% Was the trial design appropriate, and were any deviations from the standard RCT design (individual randomisation, parallel groups) accounted for in the conduct and analysis?
Was appropriate statistical analysis used?
Were outcomes measured in a reliable way?
Were outcomes measured in the same way for treatment groups?
Were participants analysed in the groups to which they were randomised?
Was follow-up complete and if not, were differences between groups in terms of their followup adequately described and analysed?
Were treatment groups treated identically other than the intervention of interest?
Were outcome assessors blind to treatment assignment?
Were those delivering treatment blind to treatment assignment?
Were participants blind to treatment assignment?
Were treatment groups similar at baseline?
Was allocation to treatment groups concealed?
Was true randomisation used for assignment of participants to treatment groups?

Discussion
The results of this systematic review indicate that there is no consensus on, or highlevel evidence for, the effectiveness of probiotic therapy for wound healing owing to the differences in the type of wounds, target population, and criteria for assessing the effect between studies. Nevertheless, it is important to highlight that none of the studies reported adverse effects due to probiotic therapy or a marked increase in the healing time of wounds.
The first studies that dealt with probiotics in dermatology, specifically in atopic dermatitis, were from the first decade of the 2000s. It is therefore an emerging theme that has mainly been researched by means of in vitro and animal studies [31][32][33].
The health-promoting properties of probiotics are suggested to be strain-dependent. The identity and characteristics of the strain are of paramount importance [34], as probiotics may regulate cytokine production and activate antimicrobial immune responses. For instance, some probiotics may induce interleukin (IL)-12, which increases the secretion of interferon (IFN)-γ and activates natural killer (NK) cells. However, they also stimulate the increase of IL-10 that induces antibody production and downregulates the inflammatory response, balancing it and contributing to healing [35]. It is noteworthy that these effects seem to be strain-specific, at least to some degree [36]. Lactobacillus strains are capable of inducing pro-inflammatory cytokines such as IL-12 and IFN-γ in addition to anti-inflammatory cytokines such as IL-10 [35], whereas Bifidobacterium strains are generally better inducers of IL-10 than Lactobacillus strains [37,38]. However, an in vitro study conducted by Dong et al. found little evidence for strain-specific effects of six probiotics on NK cell activity and NK cell or T cell activation. Cytokine production is differentially altered by the probiotic strains of distinct species. Thus, the in vivo biological importance of these strain-specific effects still needs to be elucidated [39].
The studies included in this review that found positive outcomes mainly evaluated the need for grafting, loss of graft, ulcer size, number of dressings per day, and incidence of post-operative wound complications. Meanwhile, when the wounds were assessed using clinical scores, probiotic treatment did not show any significant effect. Skin grafting is the preferred treatment for deep dermal burns, wherein necrotic and inflamed tissues are removed, and faster physiological wound closure is promoted [40]. El-Ghazely et al. found a decrease in the need for grafting in patients treated with probiotics [26]. However, because of the scarcity of studies examining the use of oral probiotics associated with grafting procedures, this effect needs further confirmation. It has already been demonstrated that the strain L. fermentum lowers keratinocyte viability and re-epithelialisation in in vitro studies [19,41]. Considering that infection is the second most common cause of graft loss [42], it is possible that therapeutic microorganisms may improve systemic immune functioning [43], favouring the healing process.
The studies included in this review did not elucidate the mechanisms of action of probiotics in the improvement of wound healing. Although the role of the intestinal microbiome in human health and disease is widely known, the role of the skin microbiome in wound healing is less well-defined [44]. Poutahidis et al. identified that oral therapy with probiotics leads to rapid deposition of collagen that is essential for proper wound healing [45]. Yu et al. assessed whether clinical data support the utility of oral and topical probiotics for certain dermatological conditions including chronic wounds. They reported that probiotics can promote the healing process by modulating the inflammatory response and limiting the colonisation of pathogens [16]. A literature review conducted by Lukic et al. identified three possible routes of action of oral probiotic therapy in wound healing. The first pathway is through the central nervous system, where probiotics produce neuroactive molecules and/or modulate the secretory activity of enteroendocrine cells in the intestinal mucosa, leading to the release of neuromodulators with the potential to improve tissue regeneration. The second route is through immunomodulation, in which intestinal probiotics can stimulate the recruitment of lymphocytes to the injured tissue, contributing to the activation of innate and adaptive immune responses. The third route is through the improved absorption of essential nutrients, especially vitamins, minerals, and enzyme cofactors involved in tissue repair to heal skin wounds [8].
Wälivaara et al. [25] and Twetman et al. [30] used the lowest doses of probiotics, in contrast to other studies [24,[26][27][28][29], and found no beneficial effects on wound healing. In addition, Wälivaara et al. [25] evaluated the effect of probiotic treatment over a longer period, and Twetman et al. [30] applied a non-validated instrument to assess wound healing. The heterogeneity of these results can also be attributed to differences in the skin microbiome between various regions of the body, such as between drier or more humid areas, or areas with a greater number of sebaceous glands [44]. Beyond this, the studies included in this review did not address the nutritional aspect and gastrointestinal microbiota of individual subjects, although it is known that these factors can influence the wound healing process, as they can affect the immune system. Both malnutrition and dysbiosis are factors that can negatively contribute to proper wound healing [3,4,46]. Therefore, it is important that future studies include the assessment of these factors in wound healing.
Regarding the age range of the patients, previous studies [1,47,48] showed that increasing age is associated with a slight delay in healing, rather than a real loss in its quality. However, it is difficult to identify whether the delay in healing is due to age, or rather diseases that are commonly associated with advancing age. Although the studies included in this review did not evaluate the age of the participants in relation to healing, it is noteworthy that Mohseni et al. found a significant improvement in ulcer healing after probiotic therapy in participants aged 40-85 years [29].
In certain diseases such as epidermolysis bullosa, or in conditions such as diabetic ulcers and extensive burns, in which the healing processes are constant or time-consuming, the need for an effective healing treatment becomes paramount in order to guarantee a better quality of life. Spanos et al. [49] evaluated the impact of the treatment of ulcers and the quality of life of patients with diabetes (n = 103) based on the following items: leisure, physical health, dependence/daily life, negative emotions and concerns about ulcers, and discomfort regarding ulcer care. After treatment, the quality of life significantly improved for all assessment items when compared with before treatment. This highlights the importance of effective healing and its relationship to quality of life as an underresearched topic. Therefore, further studies concerning the use of probiotic therapy should be encouraged, since all efforts to favour the healing process can benefit global health and quality of life, which are often compromised in patients with inflammatory skin conditions.
The studies included in this review did not report adverse effects on the clinical risk and safety of oral/enteral probiotic therapy. However, a previous report of more than 600 studies examining the safety of probiotics on microorganisms from six genera, found that, despite the actual probiotic clinical trials showing no evidence of increased risk, the present literature is not well-equipped to answer questions about probiotic safety based on intervention studies with assurance [50].
The strengths of this review are the originality of the study, as well as the methods and expert search strategies used. However, the main limitation is the low certainty of the evidence, due to the small number of studies evaluating the outcomes of interest. This may be because the use of probiotics to treat several types of wounds is an emerging topic of interest. There is also notable variation in the scales used to evaluate the effects of probiotics on wound healing, which made it impossible for us to conduct a meta-analysis.

Conclusions
We found few studies that have investigated the relationship between probiotic therapy and wound healing. Furthermore, among the studies included in this review, we observed a wide variety of probiotic strains used, types of wounds, and target populations, which prevented us from drawing clear conclusions regarding the effectiveness of probiotic therapy for wound healing. However, we found no marked increase in wound healing time or adverse effects in any of the included studies, highlighting this as a promising field for further clinical investigation.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nu14010111/s1, Table S1: Database search strategy; Table S2: Full-text articles excluded with reasons (n = 23); and Table S3: Risk of bias in the included studies-Critical Appraisal Checklist for Randomised Controlled Trials and for Quasi-Experimental Studies (non-randomised experimental studies).

Conflicts of Interest:
The authors declare no conflict of interest.